Stable Carbon Isotopic Composition of Submerged Plants Living in Karst

Aquatic Botany 140 (2017) 78–83

Contents lists available at ScienceDirect

Aquatic Botany

journal homepage: www.elsevier.com/locate/aquabot

Stable carbon isotopic composition of submerged plants living in karst water ☆ MARK and its eco-environmental importance ⁎ Pei Wanga,b,c, Gang Hua,d, Jianhua Caoa, a Karst Dynamics Laboratory (MLR and GZAR), Institute of Karst Geology, Chinese Academy of Geological Sciences, Guilin 541004, China b China University of Geosciences, Beijing 100083, China c State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China d Guilin University of Technology, Guilin 541004, China

ARTICLE INFO ABSTRACT

13 Keywords: The stable carbon isotopic composition of submerged plants (δ CP) can be controlled by physiological and Zhaidi River environmental factors. Herein, we took advantage of a short, natural karst river with an annual mean Submerged plants − −1 bicarbonate (HCO3 ) value of 3.8 mmol L to study the stable carbon isotopic composition of submerged Stable carbon isotope 13 13 plants along the river and the influence of environmental conditions on the δ CP values. The δ CP values of Dissolved inorganic carbon Ottelia acuminata, Potamogeton wrightii, Vallisneria natans, and Hydrilla verticillata from upstream to downstream Karst water environment show a gradient and ranged from −34.8‰ to −27.8‰, −36.6‰ to −23.7‰, −35.1‰ to −25.3‰, and −38.6‰ to −26.3‰, respectively and even more depleted values for the first two species at the uppermost site. Diurnal variation of water chemistry and concentration of the dissolved inorganic carbon (DIC) and the stable 13 carbon isotopic composition of DIC (δ CD) indicate that the macrophytes and other primary producers in the 13 river have a very high net photosynthetic rate. The gradient of δ CP values was consistent with CO2 being a declining source of inorganic carbon for photosynthesis in the downstream transect. The results demonstrate that the high DIC concentration with lower negative δ13C value, particularly in karst water environment has a significant role in controlling the stable carbon isotopic composition of submerged plants living in it.

1. Introduction restricted to DIC supply (Maberly and Spence, 1989). Because of the low diﬀusion rates of gases in water and the existence of a well-developed

Much recent work has focused on the inter-relationships between diffusive boundary layer around submerged plant surfaces, CO2 is the ecological, hydrological, and physico-chemical processes in ground- generally less available in water than in air. In addition, photosynthesis water/surface water interactions (Sophocleous, 2002; Hancock et al., can also be further limited by the intermittent depletion of CO2 produced 2009; Bork et al., 2009). One of the most important interactions when the rates of photosynthetic demand exceed those of replenishment between surface water and groundwater occurs in spring-fed rivers in and by the generation of high concentrations of oxygen that promotes which groundwater chemistry controls solute inputs to surface water photorespiration (Maberly and Madsen, 2002a; Pedersen et al., 2013). and represents the initial control on river ecology (Holmes, 2000; Therefore, submerged plants require high concentrations of DIC to Harvey and McCormick, 2009). Surface water function and biodiversity saturate their photosynthesis. A number of submerged macrophytes are controlled by interactions between the physical and chemical possess physiological and biochemical features that ameliorate the effect environments, in addition to the physiological and biochemical accli- of low carbon availability and minimize the effects of its potential mation and adaptation of organisms as well as their short-term limitation (Spence and Maberly, 1985). Klavsen et al. (2011) summarized behavioral responses (Maberly et al., 2015). that ‘avoidance’ and ‘exploitation’ strategies are effective approaches to

Submerged plants are important primary producers, maintaining the obtain suﬃcient CO2 for photosynthesis. In addition, ‘amelioration’ ecological balance of aquatic systems and taking part in biogeochemical strategies based on carbon dioxide concentrating mechanisms (CCMs) − cycling. However, unlike terrestrial plants, it is a common phenomenon can be present, including the ability to use HCO3 , crassulacean acid that photosynthesis and growth of aquatic primary producers are strongly metabolism (CAM) and C4-like photosynthesis (Maberly and Madsen,

☆ This article is part of a special feature entitled: “Macrophytes in freshwater habitats– Perspectives from Asia. Proceeding of the second International Symposium of Aquatic Plant Biology (MACROPHYTE 2014, Wuhan, China)” published at the journal Aquatic Botany 140C. ⁎ Corresponding author. E-mail address: [email protected] (J. Cao). http://dx.doi.org/10.1016/j.aquabot.2017.03.002 Received 1 May 2015; Received in revised form 19 December 2016; Accepted 4 March 2017 Available online 06 March 2017 0304-3770/ © 2017 Elsevier B.V. All rights reserved. P. Wang et al. Aquatic Botany 140 (2017) 78–83

− 2002b; Bowes, 2011; Dou et al., 2013). The ability of HCO3 utilization is Hydrilla verticillata are the dominant species, and these species have an thus particularly advantageous under alkaline conditions, and the most average fresh biomass of 526 ± 178 (n = 4), 357 ± 213 (n = 4), − −2 widespread CCM involves the use of HCO3 as an alternative carbon 977 ± 460 (n = 4), and 193 ± 156 (n = 4) g m , respectively. Domi- 13 source. In terrestrial plants, the δ CP value is closely related to the nant algae in Zhaidi River are Synedra sp., Navicula sp.andPinnularia sp., 13 photosynthetic pathway used in carbon fixation. The δ CP value of all belonging to Bacillariophyta,withanaveragedensityof − freshwater aquatic plant is affected by the type and extent of the CCM and (0.34 ± 0.03) × 105 (n = 3) ind. L 1. Epiphytic algae and their density also by the stable isotope signature of the carbon source and has been where unfortunately not determined during our study, although they 13 found to vary from −50‰ to −11‰ (Keeley and Sandquist, 1992). might contribute to the δ CP values of the macrophytes. At the inlet of Ecological processes, particularly the photosynthesis of macro- the Zhaidi River, the chemical composition is dominated by Ca2+ and − −1 phytes, can significantly impact the hydrochemical characteristic of HCO3 , with annual mean concentrations of 1.9 mmol L and − surface water fed by underground water (de Montety et al., 2011). 3.8 mmol L 1 respectively (Pei, 2012). The annual discharge at the outlet − Photosynthesis of macrophytes is considered to be a crucial biochemical ranges from 33 to 13000 L s 1. process in controlling the DIC diurnal cycling in spring-fed surface 13 water (Liu et al., 2008). The δ CD value has been used to improve the 2.2. Field methods understanding of the carbon cycle and diel process by macrophytes in the catchment (Heffernan and Cohen, 2010; Parker et al., 2010; Poulson Temporal variation in water chemistry was assessed at the source of 13 and Sullivan, 2010). The δ CD value in upstream regions is mainly the Zhaidi River and the point upstream from the confluence with the controlled by geochemical processes, while in the downstream, it value Chaotian River (Fig. 1C). The sampling survey started from 11:00 am on is mainly affected by photosynthesis and respiration of macrophytes September 10 and lasted to 15:00 pm on September 12, 2014. Water (Parker et al., 2007; Poulson and Sullivan, 2010). A variety of studies temperature, dissolved oxygen (DO) and pH were monitored and focused on the diurnal variation and the utilization of DIC by macro- recorded by an oxygen meter (YSI6400, YSI, USA) at 5-min interval at phytes in spring-fed rivers, particularly on the calculation of submerged the two locations. The optical DO sensor was calibrated to atmospheric macrophyte capacity for a karst carbon sink (Neal et al., 2002). oxygen concentrations before deployment and verified in the laboratory However, it is still unclear for certain submerged plants what the after deployment to be within 3% of 100% saturation. The pH sensor was 13 δ CP value is and which carbon sources they tend to use in photo- calibrated at pH 7.00 and pH 4.01 in the laboratory the day before 13 synthetic processes in karst water environment. Therefore, the δ CP deployment, the drift in pH electrodes after deployment was 0.01 pH unit. values at various sites along the karst river, along with diurnal At each site, water samples for stable carbon isotope measurement 13 variations of DIC species and concentration, as well as the δ CD values, were collected with a 100 mL disposable sterile syringe at 1-h intervals were monitored to assess the photosynthetic carbon source and karst during the daytime from 5:00 am to 8:00 pm and 3-h intervals impact on the stable carbon isotopic composition of submerged plants. overnight. Each water sample was filtered through a Millipore filter of 0.45 μm pore size and preserved in a 50 mL polyethylene bottle 2. Materials and methods without any air-space after injecting three drops of a saturated solution

of HgCl2 to prevent microbial alteration. At 6 hourly intervals, water 2.1. Site description samples were collected at the upstream and downstream site for the analysis of major water elemental components (K+,Na+,Ca2+,Mg2+, 2− − − − 2− The Zhaidi karst underground river system is located in eastern Guilin SO4 ,Cl , HCO3 ,OH , and CO3 ). The water was filtered through Haiyang village Lingchuan County, Guangxi Zhuang Autonomous Region, a Millipore filter of 0.45 μm pore size and stored in 596 mL plastic China (Fig. 1). Its geographic coordinates are 110°32′36″ to 110°37′22″ E, bottles without any air-space. The water samples were stored in 25°13′59″ to 25°18′19″ N, with approximately 32.7 km2 of recharge area. portable ice boxes until the evening when they were sent back to the The recharge area is mainly comprised of Devonian limestone, which laboratory and kept in a refrigerator at 4 °C until analysis. covers 89.5% of the total catchment. In the catchment, underground The submerged plant samples from sites A to F were collected by hand rivers, karst caves, karst sink holes, underground river skylights, and karst and washed repeatedly with a soft brush to remove adhering material and depressions are fully developed (Chen et al., 2013). Meanwhile, the main epiphytic algae from the surface of the leaves. Plant shoots were ® geomorphology is peak cluster with thin soil; scrub and grass are the transferred to Ziplock polythene bags and stored in portable ice boxes. dominant vegetation. The main land-use types are farmland and orchard in the depression, whereas in the middle and western regions, the rock 2.3. Laboratory analyses desertification is very serious. The area has a subtropical monsoon climate, hot and rainy; the annual mean temperature is about 18–19 °C, The water samples were analyzed for major cations (K+,Na+,Ca2+ and it receives an annual rainfall of about 1650 mm. The rainy season and Mg2+) with an IRIS Intrepid II XSP (Thermo Scientific, USA). The − 2− begins in April and ends in August, and this period accounts for anions Cl and SO4 were analyzed with an 861 Advanced Compact approximately 60% of the annual precipitation total. The rainfall is Ion Chromatograph (Metrohm, Switzerland). The analytical precision collected in the depression and charges the underground water through was better than 5% based on duplicate measurements of internal underground river skylights, karst funnels, karst sink-holes, karst moun- standards. Water pH was measured with SevenMulti pH meter tain foot holes. The karst aquifer medium is characterized by two (Mettler Toledo, USA) with a precision of better than 0.01 unit and − − 2− structures, enormous underground pipelines and karst fissures. The the concentration of HCO3 ,OH and CO3 in a 50 mL sample was − groundwater flows through the underground pipeline from north to south titrated within two days of collection by using 0.05 mol L 1 HCl that −1 and is discharged into the Chaotian River via the Zhaidi River, which is had been standardized against 0.05 mol L Na2CO3. The error calcu- − the site of this study (Fig. 1B). lated by averaging numerous duplicate samples was ± 0.03 mg L 1.

The Zhaidi River has a total length of 512 m and is mostly 2–6mwide The concentration of free CO2 was calculated by the geochemical and 0.6–2.2 m deep. The river has been channelized on both sides with a modeling program PHREEQC (Parkhurst and Appelo, 1999). wall. The river sediments, which range in pH value from 8.2 to 8.9, are In the laboratory, all plant samples were removed from the mainly composed of sand grains with a diameter of 0.075–2 mm, and the refrigerator, and ultrapure water (Milli-Q, Millipore, Germany) was − organic matter content is between 1.2 and 9.5 g kg 1 (unpublished data). used to carefully rinse the sample twice. Plants were then dried at The Zhaidi River is colonized by eight species of submerged plants 105 °C for 12 h to deactivate enzyme and then dried at 50 °C to a aﬃliated to four families and six genera (Wang et al., 2015). Among constant weight. Dried samples were ground in an agate mortar and them, Ottelia acuminata, Potamogeton wrightii, Vallisneria natans and passed through a 100 sieve mesh. A small amount of the powdered

79 P. Wang et al. Aquatic Botany 140 (2017) 78–83

Fig. 1. Location of the study area in China (A), showing the catchment area (dashed line), the source and the downstream ﬂow to the Zhaidi river (B) and six plants sampling sites A–F distribution in Zhaidi river (C).

Table 1 Stable carbon isotope composition of four species at diﬀerent sites.

Species A B C D E F

Ottelia acuminata −40.5‰ −34.8‰ −35.0‰ −30.6‰ −34.8‰ −27.8‰ Vallisneria natans – −35.1‰ −34.8‰. −33.3‰ −34.9‰ −25.3‰ Potamogeton wrightii −39.2‰ −36.6‰ −35.8‰ −33.1‰ −34.4‰ −23.7‰ Hydrilla verticillata – −38.6‰– −34.6‰ −34.0‰ −26.3‰ Distance (m) 0 85 128 260 332 512

“—” Species not present at the site. The distances are from the source to each sampling site.

80 P. Wang et al. Aquatic Botany 140 (2017) 78–83

− plant sample was put, together with a copper oxide wire, into a 6 mm of HCO3 and CO2, decreased during the day butroseduringthenight diameter silica tube. The tube was sealed under vacuum and combusted (Fig. 2). The diurnal change of DIC in water demonstrated that submerged for 5 h at 850 °C in a muffle furnace. Liquid N2 and dry ice were used to plants and other primary producers present utilized the DIC as carbon extract and purify the sample, and the liberated CO2 was analyzed with sources for photosynthesis, simultaneously leading to a diurnal pH change a Stable Isotope Mass Spectrometer (MAT253, Thermo Scientific, USA). of about 0.25 units (pH 7.46–7.72). The DIC together with the DO The instrument precision is better than 0.2‰ based on three replica- variation exhibited that strong photosynthesis occurred in the river. This tions. The isotope data are reported in the conventional delta notion is consistent with significant photosynthesis by the submerged plants and (‰) versus Vienna Pee Dee Belemnite (V-PDB). associated primary producers causing the diel variation of dissolved − inorganic carbon isotope in the river by consuming HCO3 and CO2. 3. Results 4. Discussion 3.1. Stable carbon isotopic composition of submerged plants 13 4.1. Influencing factors for measured δ CP values The stable carbon isotope composition of 21 plant samples belong- 13 δ13 ing to four species is shown in Table 1. The δ CP value of each species The values of CP values reported here for a karst river ranging became less depleted from upstream to downstream of the Zhaidi River. from −40.5‰ to −23.7‰ are consistent with the range reported by 13 fl δ13 The most negative δ CP value appeared at the upstream with a value of Keeley and Sandquist (1992). The two factors in uencing CP value 13 −40.5‰, and the least negative δ CP value appeared at the down- are the value of the source (complicated by whether or not CO2 or − stream with a value of −23.7‰. At the first site where all species were HCO3 was used and in what proportion) and the extent of discrimina- found (site B, 85 m from the source, Fig. 1C), there was a 3.8‰ range in tion which will depend largely on the extent of carbon limitation which 13 δ CP values and at the downstream site there was a 4.1‰ range of is controlled by the concentration of CO2 at the active site of Rubisco values among the four species. (Jasper et al., 1994; Lin and Wang, 2001). In Zhaidi River, the δ13C value of the carbon source seems to be the reason that led to a gradient δ13 ff 3.2. DIC species and concentrations in CP values for a certain species at di erent sites. Although our study is based only on each one replicate per site and species, the ff δ13 The discharge of the spring-fed river was moderate and stable during di erences in CP value between up- and downstream were large, − the monitoring periods, with a flow of 130 L s 1. At the upstream site, the supporting our conclusions on differences in the carbon source. In this δ13 δ13 − ‰ mean temperature and pH were respectively 1.7 °C and 0.1 lower than at study, the CCO2 and CHCO3- are respectively 22.2 and the downstream site. At both sites, the main cations and anions were −13.7‰ at the upstream and −21.7‰ and −13.4‰ at the down- 2+ − − Ca and HCO3 , and DIC consisted of HCO3 and CO2 (Tables 2a and stream sites, calculated after Mook et al. (1974) by using the mean − − 2− δ13 2b). At the upstream site the mean concentrations of HCO3 and CO2 temperature, concentration of CO2, HCO3 and CO3 , the CD, and −1 δ13 were 3.90 and 0.24 mmol L , while at the downstream site they had Cp at both monitoring sites. From upstream to downstream, not only −1 −1 δ13 − decreased to 3.78 mmol L and 0.12 mmol L ,respectively.Conse- the C values of CO2 and HCO3 become more depleted but also the − − quently, the molar ratio of HCO3 to CO2 declined from 16 at the ratio of HCO3 /CO2 becomes larger, which may cause the variation of upstream site to 32 at the downstream site. carbon source. For terrestrial plants as well as submerged plants, CO2 is the optimal inorganic carbon for photosynthesis, however many kinds − 3.3. Diurnal variation of DIC isotopic composition of submerged plants have the ability of using HCO3 (Madsen, 1993). 13 The results here showed that at night the δ CD values were nearly the 13 same upstream and downstream, implying that the differences during the The δ CD values in the river showed a significant spatial and temporal 13 day were caused by photosynthesis and not by other processes such as variation (Fig. 2). Compared with upstream, diurnal variation of δ CD at δ13 the downstream indicated that rapid photosynthesis had taken place, degassing of CO2. The lesser depletion in CD values during the day is which was proven by the diurnal variation of the dissolved oxygen (Fig. 3). consistent with a greater photosynthetic removal of CO2 compared to − 13 The strong photosynthetic processes of submerged plants and other HCO3 since the former is more depleted in C(Mook et al., 1974). The δ13 primary producers consumed abundant inorganic carbon, which is con- downstream reduction in depletion of CP will be partly caused by this − 13 lower depletion, but this only accounts for a maximum of about 1‰ while sistent with the variation in HCO3 and CO2 concentration and δ CD.The 13 there was downstream difference of about 10‰ in δ13C in the plants. This mean δ CD value was 0.5‰ less negative at the downstream site P ff compared to −14.2‰ at the upstream site. The diurnal variation value is likely to be caused by altered reliance on di erent forms of inorganic was less than 1‰, with the highest values occurring between 3:00 p.m. carbon for the upstream vs the downstream plants. Unlike terrestrial − and 4:00 p.m. and the lowest values occurring during the night between plants, certain submerged plants, as well as some algae, may use HCO3 in 2:00 a.m. and 3:00 a.m. at which time the carbon isotope composition addition to CO2 (Allen and Spence, 1981; Maberly and Spence, 1983). were nearly the same at both sites. The DIC in the water, which consisted Previous studies have shown that Ottelia acuminata, Potamogeton wrightii,

Table 2a Main physico-chemical conditions at site A.

+ + 2+ 2+ − 2− − Time Temperature pH K Na Ca Mg Cl SO4 HCO3 CO2

11:18 19.85 7.22 0.80 1.00 74.36 3.98 2.42 9.26 232.75 16.72 17:18 19.84 7.43 0.86 0.99 73.96 3.97 2.42 9.25 238.96 10.56 23:18 19.83 7.44 0.82 1.00 74.45 3.99 2.42 9.26 234.31 10.12 5:18 19.83 7.45 0.80 1.03 75.5 4.07 2.42 9.22 245.17 10.12 11:18 19.83 7.45 0.83 1.01 74.84 4.06 2.45 9.24 232.76 9.68 17:18 19.82 7.45 0.81 1.01 73.57 4.00 2.42 9.15 235.86 9.68 23:18 19.82 7.45 0.82 1.02 74.18 4.05 2.44 9.19 237.41 9.68 5:18 19.81 7.45 0.76 1.15 81.59 4.26 2.43 9.29 235.86 9.68 11:18 19.82 7.46 0.81 1.04 77.99 4.06 2.54 9.26 238.96 9.68 15:18 19.82 7.46 0.82 1.02 77.30 4.04 2.48 9.29 246.72 10.12

−1 In the Table, the unit for Temperature is °C, for cations, anions and CO2 the units are mg L .

81 P. Wang et al. Aquatic Botany 140 (2017) 78–83

Table 2b Main physico-chemical conditions at site F.

+ + 2+ 2+ − 2− − Time Temperature pH K Na Ca Mg Cl SO4 HCO3 CO2

11:18 21.32 7.46 0.86 1.01 75.91 4.05 2.4 9.03 237.41 9.24 17:18 22.05 7.64 0.83 1.01 75.00 4.04 2.41 9.20 229.65 6.16 23:18 21.05 7.47 0.97 1.06 76.59 4.09 2.46 9.18 232.75 9.24 5:18 20.91 7.47 0.83 1.01 76.52 4.08 2.39 9.11 234.31 9.24 11:18 21.41 7.58 0.83 1.00 74.41 4.00 2.43 9.15 226.55 7.04 17:18 22.35 7.68 0.81 1.02 73.91 4.02 2.42 9.09 223.44 5.72 23:18 21.03 7.49 0.81 1.04 74.52 4.02 2.45 9.10 235.86 9.24 5:18 20.89 7.48 0.8 1.01 74.96 4.06 2.42 9.20 234.31 9.24 11:18 21.41 7.58 0.83 1.02 73.49 3.99 2.43 9.21 232.76 7.04 15:18 22.70 7.72 0.81 1.02 73.18 3.97 2.42 9.20 221.27 5.28

−1 In the Table, the unit for Temperature is °C, for cations, anions and CO2 the units are mg L .

− 13 Fig. 2. Diel variation of concentrations of HCO3 and CO2, and of the δ C value of Fig. 3. Diel variation of temperature, pH and dissolved oxygen at the upstream (solid dissolved inorganic carbon at the upstream (circle) and downstream (triangle) sites. Night line) and downstream (dashed line) sites. Night time is shown by hatched shading. time is shown by hatched shading. − values of the four species can be explained by the utilization of HCO3 as Vallisneria natans,andHydrilla verticillata have the ability to utilize both a carbon source in a karst water environment. − HCO3 and CO2 as carbon sources (Prins et al., 1979; Prins et al., 1980; Bowes, 2011; Dou et al., 2013; Zhang et al., 2014). The reduced depletion − 4.2. Environmental importance is consistent with a greater reliance on HCO3 at the downstream site where the concentrations of CO2 are lower during the day. It is also − 2− possible that the more depleted values downstream result from carbon The CO2-HCO3 -CO3 system in water may be described by the limitation. Madsen and Maberly (1991) found carbon limitation at similar equation: − − CO2 concentrations to those reported here in macrophytes from a Danish CO (aq)+H O ⇔ HCO +H+ ⇔ CO 2 +H+ 13 2 2 3 3 stream. A similar downstream change in δ CP was reported down a − longer transect at a French karst system, although in this case it was which establishes an equilibrium mixture of H2CO3, HCO3 , and 2− caused by changing species composition away from species dependent on CO3 that make up the DIC fraction. At a pH between 7 and 9, − − CO2 near the source to species able to use both CO2 and HCO3 approximately 95% of the carbon in the water is in the form of HCO3 , 2− downstream (Maberly et al., 2015). In non-karst water, the carbon and at a pH higher than 10.1, CO3 predominates (Dreybrodt, 1988; Li isotopic composition of submerged plants ranged from −27.9‰ to and Yin, 2008; Manahan, 2000; Stumm and Morgan, 2012). In our − approximately −17.2‰ with −22.3‰ for Vallisneria natans and study, 95.7% of DIC existed in the form of HCO3 and the dissolved −17.2‰ for Hydrilla verticillata, which is less negative than in this study CO2 only contributed 4.3% at the upstream site. Downstream, the 13 − (Huang et al., 2003). Therefore, we suggest that in Zhaidi River the δ CP proportions of HCO3 and CO2 were 97.8% and 2.2%, respectively. In the karst catchment, limestone dissolution can remove atmo-

82 P. Wang et al. Aquatic Botany 140 (2017) 78–83

− Huang, L., Wu, Y., Zhang, J., Li, W., Zhou, J.Z., 2003. Distribution of C, N, P and δ13Cin sphere/soil CO2, directly giving rise to high HCO3 concentrations, – −1 aquatic plants of some lakes in the middle Yangtze valley. Acta Geosci. Sin. 24, generally 3 5 mmol L , which are several-fold higher than in non- 515–518 (in Chinese, with English abstract). karst water (Cao et al., 2011, 2012), and the alkalinity is consequently Jasper, J.P., Hayes, J.M., Mix, A.C., Prahl, F.G., 1994. Photosynthetic fraction of 13C and fi higher in karst water (Gu et al., 2009). Assuming no input of additional concentrations of dissolved CO2 in the central equatorial paci c during last 255,000 years. Paleoceanography 9, 781–798. CO2, more plant biomass is produced at higher alkalinity for the same Keeley, J.E., Sandquist, D.R., 1992. Carbon: freshwater plants. Plant Cell Environ. 15, change in pH, whereas less is produced at lower alkalinity (Stumm and 1021–1035. ff Klavsen, S.K., Madsen, T.V., Maberly, S.C., 2011. Crassulacean acid metabolism in the Morgan, 2012), which was proven by the fertilization e ect of karst context of other carbon-concentrating mechanisms in freshwater plants: a review. water on the growth of aquatic plants (Liu et al., 2010; Wang et al., Photosynth. Res. 109, 269–279. 2014). In north central Florida (USA), the decrease of DIC in Inche- Li, W., Yin, L.Y., 2008. The electrochemical methods measuring photosynthesis of submerged macrophytes. J. Wuhan Botan. Res. 26, 99–103 (in Chinese, with English tucknee River, which is a karst river, indicates that photosynthesis is the abstract). primary control on DIC variation (de Montety et al., 2011). Further- Lin, Q., Wang, S.L., 2001. The composition of stable carbon isotope and some influencing − factors of submerged plant. Acta Ecol. Sin. 21, 806 (in Chinese, with English more, it has been found that high HCO3 concentration stimulates the abstract). shoot tillering and root growth of Hydrilla verticillata and Myriophyllum Liu, Z., Liu, X., Liao, C., 2008. Daytime deposition and nighttime dissolution of calcium spicatum in both laboratory experimentation and field surveys. These carbonate controlled by submerged plants in a karst spring-fed pool: insights from high time-resolution monitoring of physico-chemistry of water. Environ. Geol. 55, characteristics are of vital importance for submerged plants to anchor 1159–1168. roots in the karst stream in which sediment mainly consists of sand with Liu, Z.H., Dreybrodt, W., Wang, H.J., 2010. A new direction in effective accounting for a diameter of 0.075–2 mm (unpublished data). Besides that, the δ13C the atmospheric CO2 budget Considering the combined action of carbonate CO2 dissolution, the global water cycle and photosynthetic uptake of DIC by aquatic values in epikarst vary within an upper limit (−22.3‰) corresponding organisms. Earth-Sci. Rev. 99, 162–172. to winter values and a lower limit (−24.2‰) corresponding to summer Maberly, S.C., Madsen, T.V., 2002a. Use of bicarbonate ions as a source of carbon in − ‰ photosynthesis by Callitriche hermaphroditica. Aquat. Bot. 73, 1–7. values for an average of 23.4 (Peyraube et al., 2013). As a Maberly, S.C., Madsen, T.V., 2002b. Freshwater angiosperm carbon concentrating consequence, CO2 used by submerged macrophytes and other primary mechanisms: processes and patterns. Funct. Plant Biol. 29, 393–405. δ13 Maberly, S.C., Spence, D.H., 1989. Photosynthesis and photorespiration in freshwater producers in karst water will lead to more negative CP values, – δ13 organisms: amphibious plants. Aquat. Bot. 34, 267 286. another important factor in controlling the CP values. Maberly, S.C., Berthelot, S.A., Stott, A.W., Gontero, B., 2015. Adaptation by macrophytes to inorganic carbon down a river with naturally variable concentrations of CO2. J. Plant Physiol. 172, 120–170. Acknowledgements Madsen, T.V., Maberly, S.C., 1991. Diurnal variation in light and carbon limitation of photosynthesis by two species of submerged freshwater macrophyte with a We acknowledge Tu Chunyan, Yin Weilu, Liu Zhiling and Guo differential ability to use bicarbonate. Freshw. Biol. 26, 175–187. Madsen, T.V., 1993. Growth and photosynthetic acclimation by Ranunculus aquatilis L. in Yongli for their valuable help in the day and night monitoring as well as response to inorganic carbon availability. New Phytol. 125, 707–715. Yang Hui for her support in the laboratory analysis. This work received Manahan, S.E., Manahan, S.E., 2000. Environmental Chemistry, seventh edition. CRC financial support from the Chinese Geological Survey Project Press LLC, Boca Raton. Mook, W.G., Bommerson, J.C., Staverman, W.H., 1974. Carbon isotope fraction between (12120113005300), Guangxi Science and Technology Development dissolved bicarbonate and gaseous carbon dioxide. Earth Planet. Sci. Lett. 22, Program (14125008-2-1), Geological Exploration projects of 169–176. Neal, C., Watts, C., Williams, R.J., Neal, M., Hill, L., Wickham, H., 2002. Diurnal and Shandong Province (2016-79) and the Karst Dynamics Laboratory, longer term patterns in carbon dioxide and calcite saturation for the River Kennet, MLR and GZAR (14-B-04) and (14-B-03). south-eastern England. Sci. Total Environ. 282, 205–231. Parker, S.R., Gammons, C.H., Poulson, S.R., DeGrandpre, M.D., 2007. Diel variations in stream chemistry and isotopic composition of dissolved inorganic carbon upper Clark References Fork River, Montana, USA. Appl. Geochem. 22, 1329–1343. Parker, S.R., Gammons, C.H., Poulson, S.R., DeGrandpre, M.D., Weyer, C.L., Smith, M.G., Babcock, J.N., Oba, Y., 2010. Diel behavior of stable isotopes of dissolved oxygen and Allen, E.D., Spence, D.H.N., 1981. The differential ability of aquatic plants to utilize the dissolved inorganic carbon in rivers over a range of trophic conditions, and in a inorganic carbon supply in fresh waters. New Phytol. 87, 269–283. mesocosm experiment. Chem. Geol. 269, 22–32. Bork, J., Berkhoff, S.E., Bork, S., Hahn, H.J., 2009. Using subsurface metazoan fauna to Parkhurst, D.L., Appelo, C.A.J., 1999. User’s guide to PHREEQC (Version 2): a computer indicate groundwater-surface water interactions in the Nakdong River floodplain, program for speciation, batch-reaction, one-dimensional transport, and inverse South Korea. Hydrogeol. J. 17, 61–75. geochemical calculations. Water-Resources Investigations Report 99–4259. U.S. Bowes, G., 2011. Single-Cell C photosynthesis in aquatic plants. chapter 5. In: 4 Geological Survey: Earth Science Information Center, Denver, Colorado, USA. Raghavendra, A.S., Sage, R.F. (Eds.), C Photosynthesis and Related CO 4 2 Pedersen, O., Colmer, T.D., Sand-Jensen, K., 2013. Underwater photosynthesis of Concentrating Mechanisms. Springer, Netherlands, Berlin, pp. 63–80. submerged plants-recent advances and methods. Front. Plant Sci. 4, 1–19. Cao, J.H., Yang, H., Kang, Z.Q., 2011. Preliminary regional estimation of carbon sink flux Pei, J.G., 2012. The Analysis of Water Quality Trends and Carbon Sinks Flux in Zhaidi by carbonate rock corrosion: a case study of the Pearl River Basin. Chin. Sci. Bull. 56, Underground River Systems. China University of Geosciences, Beijing Ph.D. 3766–3773. Dissertation (in Chinese, with English abstract). Cao, J.H., Yuan, D.X., Chris, G., Huang, F., Yang, H., 2012. Carbon fluxes and sinks: the Peyraube, N., Lastennet, R., Denis, A., Malaurent, P., 2013. Estimation of epikarst air P consumption of atmospheric and soil CO2 by carbonate rock dissolution. Acta Geol. CO2 using measurements of water δ13C , cave air P and δ13C . Geochim. Sin. 86, 963–972. TDIC CO2 CO2 Cosmochim. Acta 118, 1–17. Chen, Y.D., Cheng, Y.P., Wang, H., Jiang, Y.P., Huang, Y.Q., 2013. Quantitative tracing Poulson, S.R., Sullivan, A.B., 2010. Assessment of diel chemical and isotopic techniques to study of hydraulic and geometric parameters of a karst underground river: investigate biogeochemical cycles in the upper Klamath River Oregon, USA. Chem. exemplified by the Zhaidi underground river in Guilin. Hydrogeol. Eng. Geol. 5, Geol. 269, 3–11. 11–15 (in Chinese, with English abstract). Prins, H.B.A., Snel, J.F.H., Helder, R.J., Zanstra, P.E., 1979. Photosynthetic bicarbonate de Montety, V., Martin, J.B., Cohen, M.J., Foster, C., Kurz, M.J., 2011. Influence of diel utilization in the aquatic angiosperms Potamogeton and Elodea. Hydrobiol. Bull. 13, biogeochemical cycles on carbonate equilibrium in a karst river. Chem. Geol. 283, 106–111. 31–43. − Prins, H.B., Snel, J.F., Helder, R.J., Zanstra, P.E., 1980. Photosynthetic HCO3 utilization Dou, Y., Wang, B., Chen, L., Yin, D., 2013. Alleviating versus stimulating effects of − and OH excretion in aquatic angiosperms-light-induced pH changes at the leaf bicarbonate on the growth of Vallisneria natans under ammonia stress. Environ. Sci. surface. Plant Physiol. 66, 818–822. Pollut. Res. 20, 5281–5288. Sophocleous, M., 2002. Interactions between groundwater and surface water: the state of Dreybrodt, W., 1988. Processes in Karst Systems. Springer, Berlin. the science. Hydrogeol. J. 10, 52–67. Gu, S.P., Yin, L.Y., Li, J.L., Li, W., 2009. Diurnal CO2 exchange rates of the aquatic Spence, D.H., Maberly, S.C., 1985. Occurrence and ecological importance of HCO3- use crassulacean acid metabolism plant Isotopes sinensis Palmer at different alkalinities. among aquatic higher plants. Am. Soc. Plant Biol. 125–143. Chin. J. Plant Ecol. 33, 1184–1190 (in Chinese, with English abstract). Stumm, W., Morgan, J.J., Stumm, W., Morgan, J.J., 2012. Aquatic Chemistry: Chemical Hancock, P.J., Hunt, R.J., Boulton, A.J., 2009. Preface: hydrogeoecology, the Equilibria and Rates in Natural Waters. John Wiley and Sons, New York. interdisciplinary study of groundwater dependent ecosystems. Hydrogeol. J. 17, 1–3. Wang, P., Hu, Q.J., Yang, H., Cao, J.H., Li, L., Liang, Y., Wang, K.R., 2014. Preliminary Harvey, J.W., McCormick, P.V., 2009. Groundwater’s significance to changing hydrology, study on the utilization of Ca2+ and HCO3- in karst water by different sources of water chemistry, and biological communities of a floodplain ecosystem Everglades, Chlorella vulgaris. Carbonate Evaporite 29, 203–210. South Florida, USA. Hydrogeol. J. 17, 185–201. Wang, P., Hu, Q.J., Wang, P.H., Li, B., Cao, J.H., 2015. Effect of karst geology on the Heffernan, J.B., Cohen, M.J., 2010. Direct and indirect coupling of primary production submerged macrophyte community in Zhaidi River. Guilin. J. Hydroecol. 36, 34–39 and diel nitrate dynamics in a subtropical spring-fed river. Limnol. Oceanogr. 55, (in Chinese, with English Abstract). 677–688. Zhang, Y.Z., Yin, L.Y., Jiang, H.S., Li, W., Gontero, B., Maberly, S.C., 2014. Biochemical Holmes, R.M., 2000. 5-The importance of ground water to stream ecosystem function. In: and biophysical CO2 concentrating mechanisms in two species of freshwater Jones, J.B., Mulholland, P.J. (Eds.), Streams and Ground Waters. Elsevier, macrophyte within the genus Ottelia (Hydrocharitaceae). Photosynth. Res. 121, Amsterdam, pp. 137–148. 285–297.